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1.
J Biol Chem ; 290(32): 19681-96, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26055709

RESUMO

2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.


Assuntos
2',5'-Oligoadenilato Sintetase/imunologia , Antivirais/farmacologia , Endorribonucleases/imunologia , Exorribonucleases/química , Imunidade Inata , Bibliotecas de Moléculas Pequenas/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , Nucleotídeos de Adenina/imunologia , Nucleotídeos de Adenina/metabolismo , Antivirais/síntese química , Cristalografia por Raios X , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/metabolismo , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Exorribonucleases/imunologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Humanos , Interferon-alfa/farmacologia , Modelos Moleculares , Oligorribonucleotídeos/imunologia , Oligorribonucleotídeos/metabolismo , Poli I-C/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/metabolismo , Rhinovirus/genética , Rhinovirus/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade
2.
Antimicrob Agents Chemother ; 59(6): 3450-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25824229

RESUMO

To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 µM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets.


Assuntos
Hepacivirus/genética , Replicon/genética , Linhagem Celular , Genótipo , Humanos , Replicação Viral/genética , Replicação Viral/fisiologia
3.
Proc Natl Acad Sci U S A ; 105(8): 2773-8, 2008 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-18287036

RESUMO

Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.


Assuntos
Alcinos/metabolismo , Compostos de Anilina/metabolismo , Receptores ErbB/metabolismo , Modelos Moleculares , Pirimidinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Feminino , Isatina/análogos & derivados , Isatina/metabolismo , Espectrometria de Massas , Camundongos , Camundongos SCID , Estrutura Molecular , Pirimidinas/toxicidade , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Bioorg Med Chem Lett ; 19(5): 1332-6, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19208477
6.
Bioorg Med Chem Lett ; 19(6): 1694-7, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19237286
7.
Bioorg Med Chem Lett ; 19(1): 21-6, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19028424

RESUMO

A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.


Assuntos
Antineoplásicos/química , Receptores ErbB/antagonistas & inibidores , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Administração Oral , Animais , Camundongos , Farmacocinética , Pirimidinas/síntese química , Pirrolidinas/síntese química , Relação Estrutura-Atividade
8.
Org Lett ; 4(15): 2517-9, 2002 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-12123365

RESUMO

[structure: see text] The tetraacetylenic compound, (S)-minquartynoic acid (1), is synthesized in seven linear steps and 17% overall yield from commercially available azelaic acid monomethyl ester. The key step is a one-pot three-component Cadiot-Chodkiewicz reaction to construct the tetrayne unit without using either a diyne or a triyne intermediate.


Assuntos
Alcinos/síntese química , Fármacos Anti-HIV/síntese química , Antineoplásicos/síntese química , Ácidos Graxos Insaturados/síntese química , Alcinos/química , Anti-Helmínticos/síntese química , Ácidos Dicarboxílicos/química , Humanos , Plantas Medicinais/química , Poli-Inos , Estereoisomerismo
9.
ACS Med Chem Lett ; 3(7): 565-9, 2012 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-24900511

RESUMO

A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.

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